The mechanism generally responsible for cellular replication is DNA transcription whereby genetic information about an organism as embodied in the DNA is transferred to each subsequently formed cell. It is during this replicating process that errors in the genetic code, in the form of miscoding or base pair errors (base lesions), may be passed from one cell to further generations. Some such lesions, or groups thereof, are detrimental and may result in death of the organism or neoplasia. Other lesions may have little detectible effect or may be readily repaired by enzymes produced by or introduced into the organism. Still other base lesions are beneficial and promote diversity and adaptation. It is the first category which is of great interest to the human population.
Because destruction or alteration of DNA sequences can have catastrophic consequences, e.g. cancer, much research has been conducted into identifying why and how such alterations take place. It has been shown that exposure to high levels of radiation or oxidizing chemicals causes destruction of DNA to varying degrees. For example, it has been shown that DNA misreplication occurred when bacteria was exposed in vitro to high levels of radiation so as to cause oxidatively modified nucleotide base lesions to appear. As a consequence of this and other research, it has been proposed that a primary promoter of DNA misreplication is the formation of oxidatively modified nucleotide bases. Experiments conducted by the inventor have extended this concept to show that such modifications to DNA actually occurred in vivo. Thus, it has been established that exposure to oxidative molecules in general and oxygen radicals in particular induced DNA lesions in the form of oxidatively modified nucleotide bases, and that these lesions were linked to carcinogenesis.
Further experiments conducted by the inventor have shown that exposure to known environmental toxins also caused oxidatively modified nucleotide bases to appear in vivo. In these further experiments, feral fish were taken from an environment known to contain carcinogenic compounds. Assays were conducted for DNA lesions and the resulting data showed that high levels of certain oxidatively modified nucleotide bases were present in cancerous tissues. The data indicated that cancerous tissues contained abnormally high levels of modified purine bases: 4,6-diamino-5-formamidopyrimidine (Fapy-A) and 2,6-diamino-4-hydroxy-5-formamidopyrimidine (Fapy-G), and 8-hydroxyguanine (8-OH-Gua) and 8-hydroxyadenine (8-OH-Ade). From this research, the initiator of these modifications was identified--the hydroxyl radical (.OH).
From the foregoing findings, a determination of a cancerous or precancerous state could be made by assaying for elevated concentration levels of the aforementioned oxidatively modified nucleotide bases. This discovery is the subject of pending U.S. patent application Ser. No. 07/806,487 which is incorporated herein by reference. The significance of this discovery is that for the first time, it was shown that a broad-based indicator of a cancerous state or elevated cancer risk could be utilized by conducting an assay for abnormally high concentration levels of oxidatively modified nucleotide bases. Moreover, by identifying the .OH radical as the molecule responsible for most oxidative modifications of DNA which produced genotoxic lesions, treatments could be carried out to reduce or eliminate its presence, thereby decreasing the risk of cancer or possibly arresting its continuation.
However, the data providing the association between the presence of oxidatively modified nucleotide bases and cancer raised some new questions. For example, some feral fish obtained from a tumor bearing population were found to have elevated concentrations of the identified oxidatively modified nucleotide bases, but these fish did not exhibit tumorigenesis. While the evidence indicated that these fish were at an increased risk of tumorigenesis as compared to a normal, non-tumor bearing population, the fact remained that these fish did not exhibit tumor formation. In other words, the degree of risk was not known and the reason for the cancer non-expression was not apparent.
It is well known that certain organisms exhibit tumorigenesis even though they are not exposed to environmental influences that promote cancerous growth. A potential explanation for this phenomenon is that in aerobic eukaryotic cells, molecular oxygen is reduced and yields, in minor but detectable quantities, toxic oxygen intermediates. The intermediates are generally superoxide anions (O.sub.2.sup.-.) and hydrogen peroxide (H.sub.2 O.sub.2) which are considered undesirable, but not in and of themselves especially deleterious, and hydroxyl radicals (.OH) and singlet oxygen (O.sub.2 .uparw.) which are extremely reactive and toxic to most molecules in living cells. While O.sub.2.sup.-. and H.sub.2 O.sub.2 are not intrinsically as toxic as .OH and O.sub.2 .uparw., additional metabolic processes cause the formation of the more damaging variety: O.sub.2.sup.-.+H.sub.2 O.sub.2 .fwdarw..OH+OH.sup.- +O.sub.2. And while this reaction proceeds at a generally slow pace, metal ions such as Fe (II) that are present in most biological organisms catalyze the reaction so that the reaction rate becomes biologically significant. For a more detailed discussion of oxygen radicals, see Troll and Wiesner, Ann. Rev. Pharm. Tox, 25:509-28, 1985. Thus, the presence of any of these oxygen species is considered undesirable. Yet the penultimate question remains unanswered: Why do some organisms, ostensibly similar and existing in a similar environment, exhibit tumorigenesis while others do not?
A partial answer to the dichotomy concerning the manifestation or lack thereof of cancer in a tumor bearing population or the converse proposition may be found, in part, by the natural biological processes of aerobic eukaryotic organisms. These organisms have developed various defenses to the toxic, oxygen intermediates. Cellular production of enzymes that catalytically scavenge these intermediates significantly reduce their impact upon the cell. Examples of these enzymes are superoxide dismutase (SOD), catalase, and peroxidase. Some of these enzymes function with cofactors such as vitamin E, glutathione, and ascorbate. Thus, a balance occurs wherein the concentration levels of oxygen reduction intermediates, e.g. oxygen radicals, are held in check by biologically produced and/or acquired substances. Nevertheless, knowledge of this mechanism baits the question: Does an organism reduce the concentration levels of oxygen radicals to reduce its susceptibility of tumor formation, or does it process these radicals differently to mitigate their genotoxic effect on DNA?
The questions posed above delineate the problems confronted by the inventor of the present invention. A method for identifying the cause of tumor formation or non-formation would identify the precursor condition which determined whether cancer would manifest or not. Identification of this condition would provide a diagnostician with two important pieces of information. First, if the condition were conducive to carcinogenesis, then an assessment of cancer risk could be made. Second, modification of the condition, i.e., inhibiting the undesired reaction(s) attendant thereto, could block or reverse the manifestation of cancer. This modification of the precursor condition would not necessarily decrease the presence of a tumor promotor, such as the hydroxyl radical .OH, but rather would prevent the formation of genotoxic DNA lesions.